CD40 is a 50 kD cell surface glycoprotein molecule expressed on the surface of mature and immature B cells, macrophages, follicular dendritic cells, thymic epithelium, normal basal epithelium, and some tumor-derived cell lines. The CD40 molecule is a member of the TNF receptor family, and has important signaling functions leading to a variety of downstream effects in various cell types. Early studies showed that cross-linking of CD40 on the B cell surface with an antibody resulted in B cell proliferation and activation. Antibody cross linking of CD40 in the presence of IL-4 induces proliferation and class switching in vitro, B cell aggregation via LFA-1 (Gordon et al., 1988, J. Immunol. 140: 1425), and serine/threonine and tyrosine phosphorylation of a number of intracellular substrates (Gordon et al., 1988, supra; Uckun et al., 1991, J. Biol. Chem. 266:17478). Anti-CD40 monoclonal antibodies also prime B cells to proliferate in response to agents such as PMA (Gordon et al., 1987, Eur. J. Immunol. 17: 1535) and anti-CD20 antibody (Clark & Ledbetter, 1986, Proc. Natl. Acad. Sci. U.S.A. 83: 4494).
The receptor homology of CD40 and the antibody cross-linking studies showing a central role for CD40 in B cell activation prompted the search for a natural ligand. A mutant of the Jurkat T cell line was found to constitutively activate human B cells to secrete immunoglobulin (Yellin et al., 1991, J. Immunol. 147: 3389-3395). A monoclonal antibody, termed 5c8, was raised which specifically reacted with the mutant line, but not with the parental Jurkat cell line. The 5c8 antibody immunoprecipitated a 30 kD (more accurately, 29.3 kD, 261 amino acids) cell surface polypeptide and was found to specifically inhibit the B cell helper function of the mutant cell line. (Lederman et al., 1992, J. Exp. Med., 175: 1091-1101; Lederman et al., 1992, J. Immunol. 149: 3817-3826; Lederman et al., 1993, Curr. Opin. Immunol. 5: 439-444). The 30 kD polypeptide ligand of the 5c8 antibody was termed T-BAM, for T-B-cell Activating Molecule. A second line of studies used molecular cloning techniques to identify polypeptides that specifically bind the CD40 molecule. cDNA clones for a specific ligand of CD40 were identified in a CD40 binding assay and alternately termed CD40 Ligand (CD40L), gp39, CD154, or TRAP (Graf et al., 1992, Eur. J. Immunol. 22: 3191-3194; Armitage et al., 1992, Nature 357: 80-82; and Aruffo et al., 1993, Cell 72: 291-300). Subsequently, the CD40L clone was found to have the same structure as T-BAM (Covey et al., 1994, Mol. Immunol. 31: 471-484). Human CD40L protein shows 82.8% and 77.4% identity at the nucleic acid and amino acid levels, respectively, to a similar protein isolated from murine EL4 thymoma cells. Both of these proteins are ligands for CD40 cell surface antigen expressed on resting B cells. CD40L has also been described as IMD3, a protein involved in hyper-IgM immunodeficiency syndrome.
The human gene encoding CD40L maps to chromosome Xq26.3-q27. The gene contains five exons. Deletions, point mutations and frameshift mutations clustering within a limited region of the CD40L extracellular domain have been found to be the basis of a rare X-linked immunodeficiency syndrome (Hyper-IgM immunodeficiency syndrome, HIGM1) characterized by recurrent bacterial infections, very low or absent IgG, IgA and IgE, and normal to increased IgM and IgD serum levels. Causally-related mutations have been found to consist of clustered deletions arising by splice-donor mutations with exon skipping, splice-acceptor mutations with utilization of a cryptic splice site, and deletion/insertion events with the creation of a new splice site.
CD40L is expressed on activated, but not resting CD4+ T cells, and was found to play a particularly important role in the humoral immune response, being linked to B cell proliferation, antibody and cytokine production, and cell viability. In vivo, deletion or mutation of CD40L leads to severe immunodeficiency, both in mice and in humans, characterized by hypogammaglobulinemia and T cell deficits in cell-mediated immunity (Chess, C., 2001, in Therapeutic Immunology, 2nd edition, Austen, K. F., Burakoff, S., Rosen, F. and Strom, T., eds., Blackwell Sciences, pp. 441-456). Human CD4+ T cells infected by HIV 1, which causes severe dysfunction of cellular immunity, but paradoxically results in intense polyclonal activation of B cells, do not express CD40L. Gene and cell surface expression of the CD40L by activated T cells has been shown to be depressed in a subgroup of patients with common variable immunodeficiency (CVI). Thus, inefficient signaling via CD40 may be responsible, at least in part, for the failure of B cell differentiation in these patients.
The functional consequences of CD40L binding to CD40 include, for example, a) rescuing B cells from apoptosis induced by Fas or cross-linking of IgM, b) induction of the co-stimulator molecules CD80 (B7-1) and CD86 (B7-2) which interact with CD28 and CD152 (CTLA-4) on the surface of activated T cells; c) increased expression of other cell surface activation molecules including CD23, CD54, CD95 and lymphotoxin-a; and d) inducing immunoglobulin class switching (see Chess, supra, and references 25, 44, and 47-60 cited therein). CD40L binding to CD40 also augments the antigen-presenting functions of dendritic cells, inducing maintenance of high levels of MHC class II antigens and upregulation of accessory molecules including CD58 (LFA-3). CD40L induces cytokine production and tumoricidal activity in peripheral blood monocytes. CD40L also co-stimulates the proliferation of activated T cells, and the co-stimulation is accompanied by the production of IFN-γ, TNF-α and IL2. The expression of CD40L on murine T-helper cells and CD4+ T cells is inhibited by IFN-γ, and is inhibited on T-helper-type 2 cells by TGF-β.
CD40L upregulates the expression of CD54 by cultured Hodgkin and Reed-Sternberg cells. The increased CD54 surface expression is accompanied by increased shedding of surface-bound CD54.
CD40L has also been suggested to be important in the induction of tolerance—CD80 and CD86, which are upregulated by CD40L, interact with CD28 to provide essential co-stimulation of T cells, in concert with T cell receptor activation, that results in full activation of T cells. In the absence of CD80 and CD86-triggered activation of CD28, anergy or tolerance occurs as a consequence of antigen triggering (Linsley & Ledbetter, 1993, Ann. Rev. Immunol. 11: 191-212; Jenkins et al., 1993, Curr Opin. Immunol. 5: 361-367; and Boussiotis et al., 1996, Immunol. Rev. 153: 5-26).
The CD40L/CD40 pathway has been implicated in the in vivo priming of CD8+ cytotoxic T lymphocytes (CTSs) by CD4+ T cells. As noted, CD40L expressed on the surface of activated CD4+ T cells interacts with CD40 expressed on dendritic cells, inducing the dendritic cells to express more MHC, and signaling through CD40 can replace the requirement for CD4+ T-helper cells in priming CD8+ CTL responses. Blockade of CD40L inhibits CTL priming, emphasizing the vital role of CD40L/CD40 interactions in CTL priming by helper T cells (Ridge et al., 1998, Nature 393: 474-478; Schoenberger et al., 1998, Nature 393: 480-483; Bennett et al., 1998, Nature 393: 478-480).
CD40L can also mediate functional interactions of CD4+ T cells with other cells that express CD40, such as fibroblasts, synovial cells and endothelial cells (Yellin et al., 1995, J. Leuko. Biol. 58: 209-216; Yellin et al., 1995, J. Exp. Med. 182: 1857-1864). CD40L induces the expression of CD54 (ICAM-1) and CD106 (VCAM-1) by fibroblasts, as well as increasing fibroblast IL-6, collagenase and collagen production and inducing fibroblast proliferation. Thus, CD40L/CD40 interactions may be involved in the induction of fibrosis associated with autoimmunity and immune responses.
CD40L interaction with CD40 induces endothelial cells to express CD62E (E-selectin), ICAM-1 and VCAM-1. The upregulation of these adhesion molecules may be involved in the binding of inflammatory cells to vascular endothelium and the subsequent migration of the inflammatory cells to sites of inflammation. CD40L blockade retards the migration of leukocytes through endothelial cell barriers. In animal models of autoimmunity, antibodies to CD40L interfere with the accumulation of inflammatory cells at the site of inflammation.
CD40/CD40L interactions have been implicated in diseases having an immune or autoimmune connection. Animal models of immune-related disease in which the CD40L/CD40 pathway has been demonstrated to play a role in the pathology include, for example, murine models of systemic lupus erythematosis (Lupus or SLE; see, e.g., Kalled et al., 1998, J. Immunol. 160: 2158-2165), arthritis (collagen-induced arthritis, see, e.g., Durie et al., 1993, Science 261: 1328-1330), multiple sclerosis (experimental autoimmune encephalomyelitis, EAE; see, e.g., Howard et al., 1999, J. Clin. Invest. 103: 281-290), autoimmune thyroiditis (experimental autoimmune thyroiditis, EAT; see, e.g., Caryanniotis et al., 1997, Immunology 90: 421-426), colitis (hapten-induced colitis; see, e.g., Stuber et al., 1996, J. Exp. Med. 183: 693-698), atherosclerosis and coronary artery disease (see, e.g., Mach et al., 1998, Nature 394: 200-203), and allograft rejection (see, e.g., Parker et al., 1995, Proc. Natl. Acad. Sci. U.S.A. 92: 9560-9564; Kirk et al., 1997, Proc. Natl. Acad. Sci. U.S.A. 94: 8789-8794; Larsen et al., 1996, Nature 381: 434-438 and Blazar et al., 1997, J. Immunol. 158: 29-39).
CD40L antibody trials for treatment of human immune-related diseases include studies in patients with Lupus (see, e.g., Huang et al., 2002, Arthritis Rheum. 46: 1554-1562). A phase I trial demonstrated that anti-CD40L humanized monoclonal antibody (IDEC-131) is safe and well tolerated by patients with Lupus (Davis et al., 2001, J. Rheumatol. 28: 95-101). A phase II study with the IDEC-131 antibody showed improvement in clinical symptoms, but efficacy of the drug over placebo controls was not demonstrated (Kalunian et al., 2002, Arthritis Rheum. 46: 3251-3258). In a phase II study with BG9588 anti-CD40L antibody, clinical efficacy was demonstrated, but the study was terminated due to the occurrence of thromboembolic events (Boumpas et al., 2003, Arthritis Rheum. 48: 719-727).
U.S. Pat. No. 5,474,771 (Lederman et al.) and U.S. Pat. No. 5,876,950 (Siadak et al.) disclose murine monoclonal antibodies specific for different epitopes of human gp39. WO95/06666 (Noelle & Foy) discloses murine anti-gp39 antibodies.
U.S. Pat. No. 6,328,964 (Noelle & Claassen) discloses methods for the treatment of multiple sclerosis using gp39-specific antibodies.
U.S. Pat. No. 5,747,037 (Noelle et al.), and EP0721469B1 (Ledbetter et al.) and its U.S. counterpart U.S. Pat. No. 5,869,049 disclose anti-human monoclonal (mouse) antibodies specific for gp39. U.S. Pat. No. 5,876,718 (Noelle et al.) discloses methods of inducing T cell non-responsiveness to transplanted tissues and of treating graft-versus-host disease with anti-gp39 monoclonal (mouse) antibodies. EP0742721B1 (Noelle et al.) discloses methods of inhibiting a humoral immune response to a thymus-dependent antigen that use anti-gp39 monoclonal (mouse) antibodies. U.S. Pat. No. 6,375,950 describes methods for inducing T cell unresponsiveness to donor tissue or organs in a transplant recipient through use of anti-gp39 monoclonal (murine) antibodies.
EP1005372B1 (De Boer et al.) describes methods for the selective killing of autoreactive CD40L+ T cells using anti-CD40L monoclonal (mouse) antibody-toxin fusion proteins.
U.S. Pat. No. 6,340,459 (Yellin et al.) describes the use of murine anti gp39 monoclonal antibody 5c8 for the treatment or prevention of reperfusion injury.
EP0831906B1 (Claassen et al.) describes methods for the treatment of T cell-mediated tissue destruction in autoimmune diseases such as multiple sclerosis using anti-gp39 monoclonal (mouse) antibodies. Antibodies used in therapeutic approaches in the prior art have been divalent antibodies of murine origin.
A number of smaller antigen binding fragments of naturally occurring antibodies have been identified following protease digestion. These include, for example, the “Fab fragment” (VL-CL-CH1-VH), “Fab′ fragment” (a Fab with the heavy chain hinge region) and “F(ab′)2 fragment” (a dimer of Fab′ fragments joined by the heavy chain hinge region). Recombinant methods have been used to generate even smaller antigen-binding fragments, referred to as “single chain Fv” (variable fragment) or “scFv,” consisting of VL and VH joined by a synthetic peptide linker.
While the antigen binding unit of a naturally-occurring antibody (e.g., in humans and most other mammals) is generally known to be comprised of a pair of V regions (VL/VH), camelid species express a large proportion of fully functional, highly specific antibodies that are devoid of light chain sequences. The camelid heavy chain antibodies are found as homodimers of a single heavy chain, dimerized via their constant regions. The variable domains of these camelid heavy chain antibodies are referred to as VHH domains and retain the ability, when isolated as fragments of the VH chain, to bind antigen with high specificity ((Hamers-Casterman et al., 1993, Nature 363: 446-448; Gahroudi et al., 1997, FEBS Lett. 414: 521-526). Antigen binding single VH domains have also been identified from, for example, a library of murine VH genes amplified from genomic DNA from the spleens of immunized mice and expressed in E. coli (Ward et al., 1989, Nature 341: 544-546). Ward et al. named the isolated single VH domains “dAbs,” for “domain antibodies.” The term “dAb” will refer herein to an antibody single variable domain (VH or VL) polypeptide that specifically binds antigen. A “dAb” binds antigen independently of other V domains; however, as the term is used herein, a “dAb” can be present in a homo- or heteromultimer with other VH or VL domains where the other domains are not required for antigen binding by the dAb, i.e., where the dAb binds antigen independently of the additional VH or VL domains.
Antibody single variable domains, for example, VHH, are the smallest antigen-binding antibody unit known. For use in therapy, human antibodies are preferred, primarily because they are not as likely to provoke an immune response when administered to a patient. As noted above, isolated non-camelid VH domains tend to be relatively insoluble and are often poorly expressed. Comparisons of camelid VHH with the VH domains of human antibodies reveals several key differences in the framework regions of the camelid VHH domain corresponding to the VH/VL interface of the human VH domains. Mutation of these residues of human VH3 to more closely resemble the VHH sequence (specifically Gly 44→Glu, Leu 45→Arg and Trp 47→Gly) has been performed to produce “camelized” human VH domains that retain antigen binding activity (Davies & Riechmann, 1994, FEBS Lett. 339: 285-290) yet have improved expression and solubility. (Variable domain amino acid numbering used herein is consistent with the Kabat numbering convention (Kabat et al., 1991, Sequences of Immunological Interest, 5th ed. U.S. Dept. Health & Human Services, Washington, D.C.)) WO 03/035694 (Muyldermans) reports that the Trp 103→Arg mutation improves the solubility of non-camelid VH domains. Davies & Riechmann (1995, Biotechnology N.Y. 13: 475-479) also report production of a phage-displayed repertoire of camelized human VH domains and selection of clones that bind hapten with affinities in the range of 100-400 nM, but clones selected for binding to protein antigen had weaker affinities.
While many antibodies and their derivatives are useful for diagnosis and therapy, the ideal pharmacokinetics of antibodies are often not achieved for a particular application. In order to provide improvement in the pharmacokinetics of antibody molecules, the present invention provides single domain variable region polypeptides that are linked to polymers which provide increased stability and half-life. The attachment of polymer molecules (e.g., polyethylene glycol; PEG) to proteins is well established and has been shown to modulate the pharmacokinetic properties of the modified proteins. For example, PEG modification of proteins has been shown to alter the in vivo circulating half-life, antigenicity, solubility, and resistance to proteolysis of the protein (Abuchowski et al., J. Biol. Chem. 1977, 252:3578; Nucci et al., Adv. Drug Delivery Reviews 1991, 6:133; Francis et al., Pharmaceutical Biotechnology Vol. 3 (Borchardt, R. T. ed.); and Stability of Protein Pharmaceuticals: in vivo Pathways of Degradation and Strategies for Protein Stabilization 1991 pp 235-263, Plenum, N.Y.).
Both site-specific and random PEGylation of protein molecules is known in the art (See, for example, Zalipsky and Lee, Poly(ethylene glycol) Chemistry: Biotechnical and Biomedical Applications 1992, pp 347-370, Plenum, N.Y.; Goodson and Katre, 1990, Bio/Technology, 8:343; Hershfield et al., 1991, PNAS 88:7185). More specifically, random PEGylation of antibody molecules has been described at lysine residues and thiolated derivatives (Ling and Mattiasson, 1983, Immunol. Methods 59: 327; Wilkinson et al., 1987, Immunol. Letters, 15: 17; Kitamura et al., 1991, Cancer Res. 51:4310; Delgado et al., 1996 Br. J. Cancer, 73: 175; Pedley et al., 1994, Br. J. Cancer, 70:1126).